Quinazolin-4-one/3-cyanopyridin-2-one Hybrids as Dual Inhibitors of EGFR and BRAFV600E: Design, Synthesis, and Antiproliferative Activity

A novel series of hybrid compounds comprising quinazolin-4-one and 3-cyanopyridin-2-one structures has been developed, with dual inhibitory actions on both EGFR and BRAFV600E. These hybrid compounds were tested in vitro against four different cancer cell lines. Compounds 8, 9, 18, and 19 inhibited cell proliferation significantly in the four cancer cells, with GI50 values ranging from 1.20 to 1.80 µM when compared to Doxorubicin (GI50 = 1.10 µM). Within this group of hybrids, compounds 18 and 19 exhibited substantial inhibition of EGFR and BRAFV600E. Molecular docking investigations provided confirmation that compounds 18 and 19 possess the capability to inhibit EGFR and BRAFV600E. Moreover, computational ADMET prediction indicated that most of the newly synthesized hybrids have low toxicity and minimal side effects.


Introduction
Cancer is still a heavily researched public health issue in modern civilization.Despite intensive research, it remains a significant cause of death in wealthy countries [1][2][3].Cancer's danger rests in its capacity to infiltrate and destroy normal tissues and organs, causing them to malfunction.Furthermore, cancer can spread to other body regions, complicating treatment [4][5][6].Cytotoxic therapy has long been considered the gold standard in cancer treatment [7].However, due to inconsistencies in treatment outcomes and inadequate safety, new approaches to cancer treatment have evolved [8][9][10].The development and widespread clinical testing of precisely targeted anti-cancer tools such as therapeutic antibodies [11], tyrosine kinase inhibitors (TKIs) [12][13][14], micro-RNA therapy [15], oncolytic viruses [16], and gene-editing treatments [17] have significantly expanded the arsenal of weapons available to combat various tumor types.
Central to these advancements is the Epidermal Growth Factor Receptor (EGFR), also known as HER1, a transmembrane receptor belonging to the ErbB family alongside HER2, HER3, and HER4 [18,19].EGFR, a pivotal tyrosine kinase receptor, plays indispensable roles in various physiological processes, including cell cycle regulation [20], differentiation [21], Central to these advancements is the Epidermal Growth Factor Receptor (EGFR), also known as HER1, a transmembrane receptor belonging to the ErbB family alongside HER2, HER3, and HER4 [18,19].EGFR, a pivotal tyrosine kinase receptor, plays indispensable roles in various physiological processes, including cell cycle regulation [20], differentiation [21], and reorganization of the cytoskeleton [22].It is frequently overexpressed in several cancer types, where it participates in activities such as cell proliferation, migration, invasion, and angiogenesis [23,24].Mutations in EGFR are frequent in diseases such as non-small cell lung cancer, head and neck cancer, and colorectal cancer [25][26][27].
Nitrogen-based heterocycle molecules are a useful source of necessary building blocks for developing novel bioactive chemicals.N-Heterocyclic components are found in over three-quarters of the medications approved by the Food and Drug Administration (FDA) [32].Quinazoline and quinazolin-4-one are crucial nitrogen-based heterocycles that have been extensively researched in numerous research activities revealing their adaptive biological effects [33,34].These chemically basic compounds possess a wide range of medicinal properties, such as anti-cancer [33,35], anti-tubercular [36], anti-inflammatory [37], and antimicrobial activities [38].Many quinazoline-based anti-cancer agents were granted FDA approval and are in clinical use for cancer management, such as Erlotinib (I) (Figure 1), demonstrating remarkable potency in inhibiting growth factor receptor tyrosine kinases, particularly targeting the EGFR receptor, and featuring quinazoline scaffolds [39].Its anti-cancer effectiveness stems from its ability to hinder intracellular phosphorylation of tyrosine kinases at the ATP binding site of the receptor, block JAK2V617F-a mutant variant of JAK2, and initiate pathways that lead to apoptotic cell death [40].Despite its notable efficacy, specificity, and favorable safety record, patients frequently acquire resistance to this treatment within 8-12 months of initiating therapy due to mutations occurring in the ATP binding site of the EGFR kinase domain [41].In our recent publication, compound (II) with a quinazolin-4-one nucleus had robust antitumor activity against four cancer cell lines, as well as significant dual EGFR and BRAF V600E inhibitory action (IC50 = 0.11 µM and 0.65 µM, respectively) (Figure 1) [33].Cyanopyridine derivatives have exhibited favorable characteristics in terms of antimicrobial [42], antioxidant [43], antibiotic [44], anti-inflammatory [45], anticonvulsant [46], as well as anti-cancer potential [47,48].Because of their potential to interact with a variety of biological targets, including tubulin [49], the survivin protein [50,51], and PIM-1 Kinase [52,53], these compounds have received much interest for their anti-cancer characteristics.In our recent publication, 3-cyanopyridone/pyrazoline hybrid III (Figure 2) exhibited impressive dual inhibitory efficacy against EGFR and BRAF V600E (IC50 = 68, 65 nM, respectively), with remarkable GI50 values of 25 nM.Like Erlotinib, compound III Cyanopyridine derivatives have exhibited favorable characteristics in terms of antimicrobial [42], antioxidant [43], antibiotic [44], anti-inflammatory [45], anticonvulsant [46], as well as anti-cancer potential [47,48].Because of their potential to interact with a variety of biological targets, including tubulin [49], the survivin protein [50,51], and PIM-1 Kinase [52,53], these compounds have received much interest for their anti-cancer characteristics.In our recent publication, 3-cyanopyridone/pyrazoline hybrid III (Figure 2) exhibited impressive dual inhibitory efficacy against EGFR and BRAF V600E (IC 50 = 68, 65 nM, respectively), with remarkable GI 50 values of 25 nM.Like Erlotinib, compound III displayed a potent inhibitor especially against both cancer cell proliferation and BRAF V600E [54].In another study, compound IV (Figure 2) demonstrated significant efficacy in inhibiting cancer cell proliferation (with a GI 50 value of 0.72 µM) and promising inhibitory potential against BRAF V600E (with an IC 50 value of 58 nM), outperforming Erlotinib (with an IC 50 value of 65 nM) [47].
Pharmaceuticals 2023, 16, 1522 3 of 23 displayed a potent inhibitor especially against both cancer cell proliferation and BRAF V600E [54].In another study, compound IV (Figure 2) demonstrated significant efficacy in inhibiting cancer cell proliferation (with a GI50 value of 0.72 µM) and promising inhibitory potential against BRAF V600E (with an IC50 value of 58 nM), outperforming Erlotinib (with an IC50 value of 65 nM) [47].A challenging issue that has arisen during EGFR inhibitor therapy is the emergence of the BRAF V600E mutation as a potential resistance mechanism [55].Inhibiting BRAF can also activate EGFR, reviving tumor growth [56][57][58].Consequently, a strategic approach involving the dual inhibition of EGFR and BRAF V600E has been employed to circumvent these complexities.Several clinical trials, including patients with metastatic colorectal cancer with the BRAF V600E mutation, revealed that the combination of BRAF V600E and EGFR inhibitors was clinically successful [55,59,60].
In our ongoing research to develop antiproliferative hybrids inspired by the biological properties of compounds I-IV, we hypothesized that these hybrids might possess dual EGFR/BRAF V600E pharmacophoric elements.As shown in Figure 3, the quinazolin-4-one moiety (as seen in compounds I and II), spacer, and 3-cyanopyridin-2-one moiety (as seen in compounds III and IV) were identified as important components.As a result, combining these pharmacophores into a single compact structure can lead to the development of potent antiproliferative drugs with EGFR/BRAF V600E inhibitory actions.This approach may provide benefits such as reduced drug resistance development and fewer associated side effects.
As a result, a novel series of quinazolin-4-one/3-cyanopyridin-2-one hybrids (7-21, Figure 3) were developed and synthesized as dual EGFR/BRAF V600E inhibitors with antiproliferative activity.The newly synthesized compounds were evaluated against a panel of four human cancer cell lines.The most potent compounds were then investigated as EGFR and BRAF V600E inhibitors.Finally, molecular docking studies were carried out to evaluate their binding mechanisms and interactions inside the active sites of molecular targets.A challenging issue that has arisen during EGFR inhibitor therapy is the emergence of the BRAF V600E mutation as a potential resistance mechanism [55].Inhibiting BRAF can also activate EGFR, reviving tumor growth [56][57][58].Consequently, a strategic approach involving the dual inhibition of EGFR and BRAF V600E has been employed to circumvent these complexities.Several clinical trials, including patients with metastatic colorectal cancer with the BRAF V600E mutation, revealed that the combination of BRAF V600E and EGFR inhibitors was clinically successful [55,59,60].
In our ongoing research to develop antiproliferative hybrids inspired by the biological properties of compounds I-IV, we hypothesized that these hybrids might possess dual EGFR/BRAF V600E pharmacophoric elements.As shown in Figure 3, the quinazolin-4-one moiety (as seen in compounds I and II), spacer, and 3-cyanopyridin-2-one moiety (as seen in compounds III and IV) were identified as important components.As a result, combining these pharmacophores into a single compact structure can lead to the development of potent antiproliferative drugs with EGFR/BRAF V600E inhibitory actions.This approach may provide benefits such as reduced drug resistance development and fewer associated side effects.

Chemistry
The reaction sequences shown in Scheme 1 were used to prepare compounds 7-21.Anthranilic acid 1 was reacted with appropriate ethyl/phenyl/allyl isothiocyanates (2a-c) As a result, a novel series of quinazolin-4-one/3-cyanopyridin-2-one hybrids (7-21, Figure 3) were developed and synthesized as dual EGFR/BRAF V600E inhibitors with antiproliferative activity.The newly synthesized compounds were evaluated against a panel of four human cancer cell lines.The most potent compounds were then investigated as EGFR and BRAF V600E inhibitors.Finally, molecular docking studies were carried out to evaluate their binding mechanisms and interactions inside the active sites of molecular targets.

Chemistry
The reaction sequences shown in Scheme 1 were used to prepare compounds 7-21.Anthranilic acid 1 was reacted with appropriate ethyl/phenyl/allyl isothiocyanates (2a-c) in refluxing ethanol in the presence of triethylamine (TEA) as a base catalyst to generate 2-mercapto-3-substituted-(3H)-quinazolin-4-ones (3a-c) [33,35].N-(4-Acetylphenyl)-2-bromoacetamide 4 was synthesized in high yield according to the previously described procedure [61] by treating p-aminoacetophenone with bromoacetyl bromide in water and methylene chloride bilayer solvent in the presence of potassium carbonate.The structure of product 4 was confirmed by the reported melting point [61].Consequently, refluxing compounds 3a-c with N-(4-acetylphenyl)-2-bromoacetamide 4 in acetonitrile in the presence of TEA for 8-12 h, and the corresponding intermediates 5a-c were prepared in 75-85% yield.The melting points of compounds 5a-c were confirmed by a previously reported study [35].The synthesis of quinazolin-4-one/3-cyano-2-pyridone hybrids 7-21 was carried out by a one-pot, four-component reaction of equal amounts of acylated quinazolin-4(3H)-one 5a-c, ethyl cyanoacetate, suitably substituted benzaldehyde, and an excess of ammonium acetate in absolute ethanol with vigorous stirring at 120-130 • C for 1.5 h to obtain final conjugates 7-21.Regarding the 1 HNMR of compound 10, it exhibited a singlet signal at δ: 4.26 ppm, illustrating the presence of methylene group of SCH 2 protons as well as the presence of a singlet signal at δ: 6.77 ppm corresponding to cyanopyridine H-5.Furthermore, the NH group of the 2-pyridone ring appeared as a singlet at δ: 12.75 ppm.In the 13  To evaluate the potential cytotoxic effects of the synthesized compounds on noncancerous cells, we utilized the human mammary gland epithelial cell line, MCF-10A [62].Compounds 7-21 were incubated with MCF-10A cells for a period of 4 days.After this incubation period, the cell survival was assessed through the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay [9].Importantly, at a concentration of 50 µM, the investigated compounds did not manifest any cytotoxic effects on MCF-10A cells.For most of the compounds tested, the cell viability remained high, surpassing 86% Table 1.

Antiproliferative Activity
The antiproliferative properties of synthesized compounds are essential in assessing their therapeutic potential.In this study, we embarked on a detailed evaluation of compounds 7-21, focusing on their ability to hinder cell proliferation across diverse cancer cell lines.The chosen cell lines represent a spectrum of human cancers, which are pancreatic cancer (Panc-1), breast cancer (MCF-7), colon cancer (HT-29), and epithelial cancer (A-549).This variety ensures that the synthesized compounds' efficacy is tested against different cancer types, giving a holistic picture of their potential use.For a rigorous assessment, the widely accepted MTT assay [63,64] was employed.Using Doxorubicin, a well-established drug, as the reference standard ensures a benchmark for the synthesized compounds, allowing a contextual understanding of their efficacy.A first glance at the results in Table 2 showcases the promising antiproliferative potential of the synthesized compounds.With GI 50 values spanning 1.40 µM to 9.40 µM, these compounds exhibited commendable inhibitory effects compared to the potent Doxorubicin with a GI 50 of 1.10 µM.Among the series, compounds 8, 9, and 18-21 emerged as the frontrunners.Their antiproliferative activity was strikingly high, especially in compound 18 with a GI 50 value tantamount to Doxorubicin, thereby indicating its impressive potency.The antiproliferative activity seems to hinge significantly on the type of substituent at the N-3 position of the quinazoline moiety.Compound 18, with an allyl group, showed a pronounced effect, whereas its counterparts with ethyl or phenyl groups, i.e., compounds 8 and 13, were less potent.This suggests the paramount role of allyl group in enhancing the antiproliferative activity, a crucial insight for future design improvements.Diving deeper into the structure-activity relationship, the position-4 substitution on the phenyl group of the pyridine-2-one moiety emerges as a critical determinant.Compounds 19, 20, and 21, bearing halogens, suggest a clear tolerance and even preference for halogens in antiproliferative activity.Interestingly, the order of efficacy based on the halogens is Cl > Br > F. Unsubstituted derivatives, namely compounds 7, 12, and 17, showcased relatively lower potency.This signifies the relevance of the substituents at position-4 of the phenyl group, stressing their importance in future design considerations.In synthesizing compounds for antiproliferative purposes, the interplay between different functional groups and substituents becomes apparent.This detailed evaluation of the synthesized compounds offers valuable insights, not just in understanding their current efficacy but also in directing future compound designs and modifications for even enhanced therapeutic potential.

EGFR Inhibitory Activity
The most effective antiproliferative hybrids, 8, 9, 18, and 19, were tested for EGFR-TK inhibitory activity as a possible target for their antiproliferative activity [65].Table 3 shows the IC 50 values for each compound against Erlotinib, the reference drug.Compounds 8, 9, 18, and 19 displayed promising EGFR inhibitory activity, with IC 50 values ranging from 110 nM to 190 nM compared to Erlotinib (IC 50 = 80 ± 5 nM).As EGFR inhibitors, the investigated compounds were less effective than Erlotinib.The most potent antiproliferative agent, compound 18 (R 1 = allyl, R 2 = OCH 3 ), was also the most potent EGFR inhibitor, with an IC 50 value of 110 ± 10 nM, 1.4-fold less potent than Erlotinib.Compound 19 (R 1 = allyl, R 2 = Cl) was the second most active compound, with an IC 50 value of 140 ± 11 nM, 1.8-fold less potent than Erlotinib.These findings add to the body of evidence supporting the significance of the methoxy group in these compounds' antiproliferative effect.Finally, compounds 8 (R 1 = ethyl, R 2 = OCH 3 ) and 9 (R 1 = ethyl, R 2 = Cl) were the least effective EGFR inhibitors, with IC 50 values of 160 ± 12 nM and 190 ± 15 nM, respectively.These findings indicate that the EGFR may be a potential target for the antiproliferative activity of the investigated compounds, notably compounds 18 and 19.

BRAF V600E Inhibitory Activity
An in vitro study was carried out to evaluate the activity of the newly synthesized compounds 8, 9, 18, and 19 against BRAF V600E [59].Table 3 shows the IC 50 values for the tested compounds and Erlotinib, which was chosen as the reference drug.Table 2 shows that the investigated compounds had good inhibitory efficacy, with IC 50 values ranging from 140 nM to 290 nM compared to Erlotinib (IC 50 = 60 ± 5 nM).Compounds 18 (R 1 = allyl, R 2 = OCH 3 ) and 19 (R 1 = allyl, R 2 = Cl) had the most potent inhibitory activities against BRAF V600E , with IC 50 values of 140 ± 10 nM and 190 ± 12 nM, respectively, being 2-and 3-fold less potent than Erlotinib.According to the findings of this study, the tested compounds 18 and 19 had dual inhibitory effects against both EGFR and BRAF V600E , suggesting that they could be used as potential targets for antiproliferative action.

Docking Study
In this investigative study, we conducted a computational docking analysis to unveil the binding interactions involving the highly in vitro active compounds 18 and 19 with the tyrosine kinase receptors EGFR and BRAF V600E .We employed the Discovery Studio software, a computational tool designed for such analyses [66][67][68].To streamline our inquiry, we obtained the X-ray crystallography structures of the EGFR and BRAF V600E tyrosine kinases.Specifically, the structure of EGFR was co-crystallized with Erlotinib as a reference structure (PDB: 1M17) [69].Similarly, the BRAF V600E structure was co-crystallized with Vemurafenib as a reference (PDB: 3OG7) [70].
To validate the reliability of the EGFR docking procedure, we conducted a re-docking experiment using the co-crystallized Erlotinib.This was performed within the active site of EGFR, resulting in a S score of −8.69 kcal/mol.The root-mean-square deviation (RMSD) value, a structural similarity measure, was calculated to be 1.72 Å.A significant hydrogen bond interaction was established, specifically between the pyrimidine nitrogen of Erlotinib and the amino acid Met769, with a bond length of 2.29 Å.This interaction emphasized the pivotal role of Met769 in stabilizing the ligand within the binding site Figure 4.

Docking Study
In this investigative study, we conducted a computational docking analysis to unveil the binding interactions involving the highly in vitro active compounds 18 and 19 with the tyrosine kinase receptors EGFR and BRAF V600E .We employed the Discovery Studio software, a computational tool designed for such analyses [66][67][68].To streamline our inquiry, we obtained the X-ray crystallography structures of the EGFR and BRAF V600E tyrosine kinases.Specifically, the structure of EGFR was co-crystallized with Erlotinib as a reference structure (PDB: 1M17) [69].Similarly, the BRAF V600E structure was co-crystallized with Vemurafenib as a reference (PDB: 3OG7) [70].
To validate the reliability of the EGFR docking procedure, we conducted a re-docking experiment using the co-crystallized Erlotinib.This was performed within the active site of EGFR, resulting in a S score of −8.69 kcal/mol.The root-mean-square deviation (RMSD) value, a structural similarity measure, was calculated to be 1.72 Å.A significant hydrogen bond interaction was established, specifically between the pyrimidine nitrogen of Erlotinib and the amino acid Met769, with a bond length of 2.29 Å.This interaction emphasized the pivotal role of Met769 in stabilizing the ligand within the binding site Figure 4.The molecular modelling and interactions presented in the docking studies of compounds 18, 19, and the quinazoline-single moiety 5C with the EGFR receptor provide a profound insight into the mechanisms and intricacies of drug-receptor binding.The The molecular modelling and interactions presented in the docking studies of compounds 18, 19, and the quinazoline-single moiety 5C with the EGFR receptor provide a profound insight into the mechanisms and intricacies of drug-receptor binding.The conjugation of quinazolin-4-one and 3-cyanopyridin-2-one motifs into a singular entity in compounds 18 and 19 and their subsequent interactions illuminate the significance of strategic molecular design for targeted receptor engagement.A critical observation from the provided data is the pronounced binding efficacy of the conjugated hybrids, 18 and 19, despite the evident absence of direct interactions of their quinazoline ring with the receptor amino acids.This absence might initially seem counterintuitive, given the potent nature of quinazoline as a pharmacophore.However, the enhanced activity of these hybrids can be attributed to their encompassing and vital interactions with pivotal EGFR amino acids.Such interactions not only anchor the molecules within the active site, but they also likely disrupt native functions of the receptor, rendering it inactive (Figure 5).The diversity and multiplicity of the interactions exhibited by compounds 18 and 19 emphasize the wisdom behind the conjugation of the two moieties.Whereas each moiety is capable of specific interactions, their combination seems to exploit a broader range of binding possibilities, ensuring a comprehensive engagement with the receptor.This intricate binding landscape, especially the interactions with crucial amino acids like MET A:769, ALA A:719, and VAL A:702, indicates a highly strategic alignment within the receptor active site, which could explain their potent in vitro activity.Compound 18 establishes a more diverse binding Pharmaceuticals 2023, 16, 1522 9 of 23 pattern within the active site of EGFR compared to compound 19.This diversity is evident in its broader interaction landscape, especially its π-sigma and π-alkyl interactions with pivotal amino acids like PHE A:771 Figure 5.These extensive interactions not only stabilize the compound within the active site but could also enhance its inhibitory effect on the receptor.Furthermore, compound 18 s computed docking score of −7.35 kcal/mol, which is higher than the −6.85 kcal/mol score of compound 19, provides a quantitative measure of its superior binding affinity (Figure 5).The constrained binding pattern of compound 19, in contrast, might limit its binding stability and, consequently, its therapeutic efficacy.Whereas compound 19 still exhibits valuable interactions and remains a potential EGFR inhibitor, compound 18, with its multifaceted engagement and superior docking score, stands out as the more potent entity in the series.On the contrary, the quinazoline-single moiety 5c, although demonstrating a series of interactions, lacks the diversity and depth of engagements witnessed in the hybrids (Figure 6).This difference is likely due to the absence of the 3-cyanopyridin-2-one motif, which, when present in the hybrids, amplifies their binding profiles.The lower S score for 5c compared to the hybrids further supports this notion (Figure 6).
sity and multiplicity of the interactions exhibited by compounds 18 and 19 emphasize the wisdom behind the conjugation of the two moieties.Whereas each moiety is capable of specific interactions, their combination seems to exploit a broader range of binding possibilities, ensuring a comprehensive engagement with the receptor.This intricate binding landscape, especially the interactions with crucial amino acids like MET A:769, ALA A:719, and VAL A:702, indicates a highly strategic alignment within the receptor active site, which could explain their potent in vitro activity.Compound 18 establishes a more diverse binding pattern within the active site of EGFR compared to compound 19.This diversity is evident in its broader interaction landscape, especially its π-sigma and π-alkyl interactions with pivotal amino acids like PHE A:771 Figure 5.These extensive interactions not only stabilize the compound within the active site but could also enhance its inhibitory effect on the receptor.Furthermore, compound 18′s computed docking score of −7.35 kcal/mol, which is higher than the −6.85 kcal/mol score of compound 19, provides a quantitative measure of its superior binding affinity (Figure 5).The constrained binding pattern of compound 19, in contrast, might limit its binding stability and, consequently, its therapeutic efficacy.Whereas compound 19 still exhibits valuable interactions and remains a potential EGFR inhibitor, compound 18, with its multifaceted engagement and superior docking score, stands out as the more potent entity in the series.On the contrary, the quinazoline-single moiety 5c, although demonstrating a series of interactions, lacks the diversity and depth of engagements witnessed in the hybrids (Figure 6).This difference is likely due to the absence of the 3-cyanopyridin-2-one motif, which, when present in the hybrids, amplifies their binding profiles.The lower S score for 5c compared to the hybrids further supports this notion (Figure 6).Similarly, to validate the docking protocol concerning BRAF V600E , we conducted a redocking experiment utilizing the co-crystallized Vemurafenib.This endeavor took place at active site of BRAF V600E , resulting in an impressive S score of −11.99 kcal/mol.To gauge the extent of structural conformity, we computed a root-mean-square deviation (RMSD) value of 1.55 Å. Substantial hydrogen bond interactions were established, particularly between Vemurafenib and the amino acids Cys532 and Asn580, firmly anchoring its position within the binding site (Figure 7).Similarly, to validate the docking protocol concerning BRAF V600E , we conducted a redocking experiment utilizing the co-crystallized Vemurafenib.This endeavor took place at active site of BRAF V600E , resulting in an impressive S score of −11.99 kcal/mol.To gauge the extent of structural conformity, we computed a root-mean-square deviation (RMSD) value of 1.55 Å. Substantial hydrogen bond interactions were established, particularly between Vemurafenib and the amino acids Cys532 and Asn580, firmly anchoring its position within the binding site (Figure 7).Similarly, to validate the docking protocol concerning BRAF V600E , we conducted a redocking experiment utilizing the co-crystallized Vemurafenib.This endeavor took place at active site of BRAF V600E , resulting in an impressive S score of −11.99 kcal/mol.To gauge the extent of structural conformity, we computed a root-mean-square deviation (RMSD) value of 1.55 Å. Substantial hydrogen bond interactions were established, particularly between Vemurafenib and the amino acids Cys532 and Asn580, firmly anchoring its position within the binding site (Figure 7).Hybrid 18, with a docking score of -7.85 kcal/mol, showcases more pronounced interactions with the receptor.From the visualization, hybrid 18 forms strong conventional hydrogen bonds with residues like CYS A:532, ASN A:580, and SER A:536 (Figure 8).These Hybrid 18, with a docking score of −7.85 kcal/mol, showcases more pronounced interactions with the receptor.From the visualization, hybrid 18 forms strong conventional hydrogen bonds with residues like CYS A:532, ASN A:580, and SER A:536 (Figure 8).These interactions, particularly with CYS A:532, are of utmost importance, considering the pivotal role cysteine residues often play in the active sites of various proteins.The compound aromatic regions also engage in multiple pi interactions, further reinforcing its grip within the active site.Alkyl and pi-alkyl interactions with residues such as ALA A:481, LYS A:483, and VAL A:471 solidify its binding affinity, potentially leading to its potent in vitro activity (Figure 8).On the other hand, hybrid 19, despite having a commendable docking score of −7.53 kcal/mol, falls slightly short when compared to hybrid 18.The visual representation suggests robust interactions, but the comparative difference in docking scores indicates that hybrid 18 might have a slightly superior binding affinity or forms more favorable interactions, translating to enhanced inhibitory potential.However, with a docking score of −6.33 kcal/mol, Erlotinib interaction is somewhat less optimal than the scores observed for hybrids 18 and 19 (Figure 8).One reason for this could be the extent and depth of interactions Erlotinib forms with the receptor.Whereas it does form important bonds with vital residues, it may not span as diverse a landscape as the hybrids, especially hybrid 18 (Figure 8).These findings closely aligned with dual EGFR/BRAF V600E in vitro inhibition assay outcomes.In conclusion, the findings presented in this study underscore the notable inhibitory potential of compound 18 against the BRAF V600E kinase.This propensity is underpinned by meaningful binding interactions observed within the active site.This observation resonates with the outcomes obtained from the rigorous BRAF V600E in vitro assay, lending credibility to the predictive capabilities of our computational approach.
18 and 19 (Figure 8).One reason for this could be the extent and depth of interactions Erlotinib forms with the receptor.Whereas it does form important bonds with vital residues, it may not span as diverse a landscape as the hybrids, especially hybrid 18 (Figure 8).These findings closely aligned with dual EGFR/BRAF V600E in vitro inhibition assay outcomes.In conclusion, the findings presented in this study underscore the notable inhibitory potential of compound 18 against the BRAF V600E kinase.This propensity is underpinned by meaningful binding interactions observed within the active site.This observation resonates with the outcomes obtained from the rigorous BRAF V600E in vitro inhibition assay, lending credibility to the predictive capabilities of our computational approach.Additionally, our investigation unveils a compelling avenue for potential dual inhibition, as indicated by supplementary simulations targeting both EGFR and BRAF V600E ; specifically, compounds 18 and 19 promise to function as dual inhibitors.
The insights garnered from this study offer a foundational comprehension of the inhibitory implications embedded within the investigated compounds.This understanding, in turn, sets the stage for subsequent experimental investigations and optimization endeavors within the dynamic realm of drug discovery.Describing the complex interactions between these compounds and the target kinases is an important guide for developing targeted hit compounds.These compounds could interfere with pathways linked to cancer signaling.Additionally, our investigation unveils a compelling avenue for potential dual inhibition, as indicated by supplementary simulations targeting both EGFR and BRAF V600E ; specifically, compounds 18 and 19 promise to function as dual inhibitors.

In Silico ADMET Studies
The insights garnered from this study offer a foundational comprehension of the inhibitory implications embedded within the investigated compounds.This understanding, in turn, sets the stage for subsequent experimental investigations and optimization endeavors within the dynamic realm of drug discovery.Describing the complex interactions between these compounds and the target kinases is an important guide for developing targeted hit compounds.These compounds could interfere with pathways linked to cancer signaling.

In Silico ADMET Studies
Considering the promising in vitro and silico docking results, we proceeded to conduct supplementary ADMET studies for the newly synthesized compounds.This choice was motivated by the intention to gain a more comprehensive insight into these significant activities [71].During the ADMET investigations, we utilized Erlotinib as the established reference compound.Using Discovery Studio 4.0, we conducted predictions for the ADMET descriptors of all synthesized compounds.The expected descriptors are outlined in Table 4.A significant number of the hybrid compounds exhibited a modest predicted level of intestinal absorption (absorption level = 2).This positioning designates them as promising candidates for localized treatment of gastrointestinal tumors or as potential contenders for intravenous administration.Notably, most of these novel compounds displayed low solubility (ADME aqueous solubility level = 2).
Furthermore, the AlogP98 tool aided in predicting the logP value based on the molecular structure of each compound.This approach allows researchers to estimate how a compound will partition between a nonpolar solvent (octanol) and water.Notably, most hybrid compounds demonstrated AlogP98 values ranging from 5 to 6, indicating their heightened lipophilicity.This characteristic contributes to their reduced aqueous solubility.However, there is a potential solution in the form of Cyclodextrin Complexation, a method that improves solubility by encapsulating these hybrid molecules within the hydrophobic cavity of cyclodextrin molecules.
During the ADMET assessment, all the newly synthesized hybrids were categorized at a blood-brain barrier (BBB) level of 4, effectively hindering their ability to penetrate across the BBB.Importantly, the drug bioavailability was interconnected with the fundamental property of 2D polar surface area (ADMET 2D PSA).
Utilizing the calculated 2D polar surface area (PSA 2D) and Atom-based Log P98 (A log P98) properties, the outcomes have been visualized through a 2D ADMET plot (Figure 9).It is worth noting that molecules with a PSA less than 145 generally exhibit characteristics of low bioavailability and passive absorption [72].Using a 2D chemical structure as input, the cytochrome P450 2D6 (CYP2D6) model predicts the potential for inhibiting the CYP2D6 enzyme.This enzyme, located in the liver, plays a critical role in metabolizing a wide range of substrates, thereby significantly contributing to various drug-drug interaction scenarios [73].As a result, experimenting to evaluate CYP2D6 inhibition becomes crucial within the regulatory protocols followed during drug discovery and development [74].Each evaluated hybrid compound was predicted to display noninhibitory behavior towards CYP2D6.As a result, the risk of inducing liver dysfunction after administering these hybrids is minimal.

In Silico Toxicity Predictions
To predict toxicity, we utilized the constructed and validated models within the Discovery Studio software for the synthesized compounds [75].The rodent carcinogenicity test carried out by the FDA evaluates the capacity of a chemical structure to induce cancer in rats.On the other hand, the prediction of rat maximum tolerated dose (MTD) estimates the highest dose of a chemical substance that can be administered to rats without resulting in adverse effects [76].In assessing toxicity for a chemical compound, the prediction of rat oral LD50 is utilized to forecast the acute median lethal dose (LD50) in rats after the compound is administered orally [77].As part of the Draize test, ocular irritancy analysis is utilized to determine the potential of a particular compound to cause eye irritation and The plasma protein binding model determines whether a substance will bind robustly (>90%) to carrier proteins in the blood.Notably, a substantial binding to plasma proteins (>90%) was expected for the majority of hybrid compounds, as detailed in Table 4.

In Silico Toxicity Predictions
To predict toxicity, we utilized the constructed and validated models within the Discovery Studio software for the synthesized compounds [75].The rodent carcinogenicity test carried out by the FDA evaluates the capacity of a chemical structure to induce cancer in rats.On the other hand, the prediction of rat maximum tolerated dose (MTD) estimates the highest dose of a chemical substance that can be administered to rats without resulting in adverse effects [76].In assessing toxicity for a chemical compound, the prediction of rat oral LD 50 is utilized to forecast the acute median lethal dose (LD 50 ) in rats after the compound is administered orally [77].As part of the Draize test, ocular irritancy analysis is utilized to determine the potential of a particular compound to cause eye irritation and assess the degree of severity of this irritation [78].In evaluations based on rabbits, investigations into skin irritancy assess the probability of a substance causing skin irritation and the potential severity of such effects.As per the in silico assessments outlined in Table 5, most compounds displayed low toxicity and minimal adverse effects.Additionally, all the hybrid compounds subjected to testing were forecasted to have non-carcinogenic attributes, aligning with the preliminary findings of the FDA rodent carcinogenicity assessment.The majority of the evaluated compounds exhibited higher rat oral LD50 values compared to the value of Erlotinib.
Furthermore, the predictive models suggested that all hybrids are anticipated to not irritate in the context of skin irritancy testing, and the majority of them are projected to induce mild irritation in the context of ocular irritancy testing.
In conclusion, the ADMET investigations conducted in this study provide crucial insights into the potential effectiveness, safety, and pharmacokinetic behavior of the newly developed hybrids.The insights gained from these assessments hold immense significance in guiding the drug discovery and development trajectory.This wealth of information assists in identifying promising drug candidates that merit further evaluation and advancement in the testing and developmental stages.
In summation, the ADMET explorations undertaken in this investigation offer pivotal insights into the potential efficacy, safety profile, and pharmacokinetic attributes of the recently formulated hybrids.The discernments gleaned from these analyses carry substantial importance, serving as a compass to navigate the path of drug discovery and development.This reservoir of knowledge aids in pinpointing auspicious drug candidates warranting meticulous scrutiny and progression through subsequent testing and developmental phases.
Finally, introducing substituents into the quinazolin-4-one and 3-cyanopyridin-2-one moieties significantly influences the efficacy of the newly synthesized hybrids 7-21.The highest levels of anti-proliferative activity were observed in 3-allyl quinazolin-4-one derivatives, with 3-ethyl and 3-phenyl derivatives displaying lower levels of activity in that order.The addition of specific functional groups, such as an electron-donating methoxy group at position 4 of the phenyl group in the 3-cyano-2-pyridone moiety (as seen in compound 18), appears to enhance both EGFR and BRAF inhibitory activity.The introduction of electron-withdrawing substituents at the para position of phenyl at position 4 of 3-cyano-2-pyridone seems to improve cytotoxicity in the order (4-Cl > 4-Br > 4-F).Moreover, the presence of a nitrile group (CN) at the C3 and carbonyl group of 3-cyano-2-pyridone moiety are essential for binding EGFR and BRAF active site.Moreover, both the pyridone nitrogen and the carbonyl oxygen atom within the 3-cyanopyrid-2-one core form hydrogen bonds with the backbone NH of Met769 located in the hinge region of the active site of EGFR kinase.On other hand, the nitrile group within the 3-cyanopyrid-2-one core is essential for forming a hydrogen bond with the key residue Cys 532 in the N-lobe, while the carbonyl oxygen atom of the amidic linkage is crucial for creating a hydrogen bond with Asn 580 within the catalytic loop active site of BRAF V600E .The strategic positioning of these functional groups is critical in defining these compounds' antiproliferative efficacy.

Chemistry
General details: see Section A (Supplementary File).Compounds 3a-c and 5a-c were prepared according to previous studies [33,35].

Chemistry
General details: see Section A (Supplementary File).Compounds 3a-c and 5a-c were prepared according to previous studies [33,35].

Figure 1 .
Figure 1.Structure of compounds I and II.

Figure 1 .
Figure 1.Structure of compounds I and II.

Figure 2 .
Figure 2. Structure of compounds III and IV.

Figure 2 .
Figure 2. Structure of compounds III and IV.
(µM): Expressed as mean ± SEM. b A-549: Human lung carcinoma cell line.c MCF-7: Human breast adenocarcinoma cell line.d Panc-1: Human pancreatic cancer cell line.e HT-29: Human colon cancer cell line.f GI 50 : average of IC 50 against four cancer cell line.

Figure 4 .
Figure 4. Docking representation model of co-crystallized Erlotinib within the binding site of EGFR (PDB ID: 1M17).

Figure 4 .
Figure 4. Docking representation model of co-crystallized Erlotinib within the binding site of EGFR (PDB ID: 1M17).

Figure 7 .
Figure 7. Docking representation model of co-crystallized Vemurafenib within the binding site of BRAF (PDB ID: 3OG7).

Figure 7 .
Figure 7. Docking representation model of co-crystallized Vemurafenib within the binding site of BRAF (PDB ID: 3OG7).

Table 5 .
The predicted toxicity properties of the synthesized compounds.
a Unit: g/kg body weight.